Mineral Apposition Rate Research Articles

Intraosseous Injection of Simvastatin in Poloxamer 407 Hydrogel Improves Pedicle-Screw Fixation in Ovariectomized Minipigs.

Osteoporosis leads to poor osseointegration and reduces implant stability. Statins have been found to stimulate bone formation, but the bioavailability from oral administration is low. Local application may be more effective at augmenting bone formation and enhancing implant stability. This study was performed to evaluate the efficacy of an intraosseous injection of simvastatin in thermosensitive poloxamer 407 hydrogel to enhance pedicle-screw fixation in calcium-restricted ovariectomized minipigs. Nine mature female Guangxi Bama minipigs underwent bilateral ovariectomy and were fed a calcium-restricted diet for 18 months. Simvastatin (0, 0.5, or 1 mg) in thermosensitive poloxamer 407 hydrogel was injected into the lumbar vertebrae (L4-L6), and titanium alloy pedicle screws were implanted. Bone mineral density (BMD) measurements of the lumbar vertebrae were determined by dual x-ray absorptiometry (DXA) before and 3 months after treatment. The lumbar vertebrae were harvested and analyzed with use of microcomputed tomography, biomechanical pull-out testing, histological analysis, and Western blot analysis for bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF) expression. Evaluation over a 3-month study period demonstrated that the BMD of the vertebrae injected with 0.5 and 1.0 mg of simvastatin had increased by 31.25% and 31.09%, respectively, compared with vehicle-only injection (p ≤ 0.00014 for both) and increased by 32.12% and 28.16%, respectively, compared with the pre-treatment levels (p < 0.0001 for both). A single injection of simvastatin in poloxamer 407 increased trabecular volume fraction, thickness, and number and decreased trabecular separation (p ≤ 0.002). The bone formation and mineral apposition rates significantly increased (p ≤ 0.023). The percentage of osseointegration in the simvastatin 0.5 and 1-mg groups was 46.54% and 42.63% greater, respectively, than that in the vehicle-only group (p ≤ 0.006), and the maximum pull-out strength was 45.75% and 51.53% greater, respectively, than in the vehicle-only group (p ≤ 0.0005). BMP-2 and VEGF expressions were higher than for the vehicle-only injection. A single intraosseous injection of simvastatin in thermosensitive poloxamer 407 hydrogel stimulated bone formation, increased BMD, improved bone microstructure, promoted osseointegration, and significantly enhanced the stability of pedicle screws in calcium-restricted ovariectomized minipigs. These results provide rationale for evaluating intraosseous injection of simvastatin in poloxamer 407 to enhance implant fixation in patients with osteoporosis.

Effect of simvastatin on osteogenesis of the lumbar vertebrae in ovariectomized rats.

The aim of the present study was to assess the role of simvastatin on osteoporosis of the vertebrae by examining the effect of simvastatin on the osteogenesis of the lumbar vertebra in ovariectomized (OVX) rats. A total of 60 6-month-old female Sprague Dawley rats were divided into one sham group and five ovariectomized groups, consisting of four simvastatin groups and one control group. Four dosages of simvastatin (5, 10, 20 and 40 mg/kg/d) were administered by gavage for three months. L4 vertebrae were examined by dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT) to determine the mineral apposition rate (MAR). L5 vertebrae were examined using a compression biomechanical test. Although the measurements from DEXA, pQCT and MAR, and the biomechanical parameters in the OVX + simvastatin rats were higher than those for the OVX + vehicle group, no significant differences were detected. Therefore, simvastatin may not improve osteogenesis of the lumbar vertebra in OVX rats or prevent osteoporosis of the spinal vertebrae.

Effects of abaloparatide-SC (BA058) on bone histology and histomorphometry: The ACTIVE phase 3 trial

There are a number of effective treatments for osteoporosis but most are in the antiresorptive class of compounds. Abaloparatide-SC is a new osteoanabolic agent, which increased bone mineral density and lowered the risk of osteoporosis-related fractures in the phase 3 ACTIVE trial. The objective of this report is to describe the effects of abaloparatide-SC 80μg on bone histology and histomorphometry in iliac crest bone biopsies from this trial in which participants were randomized to receive blinded daily subcutaneous injections of placebo or abaloparatide-SC 80μg/d or open-label teriparatide 20μg/d for 18months. Iliac crest bone biopsies were obtained between 12 and 18months. Qualitative histological analysis of biopsies from abaloparatide-SC-treated patients revealed normal bone microarchitecture without evidence of adverse effects on mineralization or on the formation of normal lamellar bone. There were no bone marrow abnormalities, marrow fibrosis nor was there presence of excess osteoid or woven bone. There were few significant differences among the three treatment groups in a standard panel of static and dynamic histomorphometric indices. The mineral apposition rate was higher in the teriparatide-treated group than in the placebo-treated group. The eroded surface was lower in the abaloparatide-SC-treated group than in the placebo-treated group. Cortical porosity was higher in both the abaloparatide-SC- and the teriparatide-treated groups than in the placebo-treated group. We conclude that histological and histomorphometric analysis of iliac crest bone biopsies from subjects who were treated for up to 18months with abaloparatide-SC showed no evidence of concern for bone safety.Trial registration: ClinicalTrials.gov number NCT01343004.

Incorporating simvastatin/poloxamer 407 hydrogel into 3D-printed porous Ti6Al4V scaffolds for the promotion of angiogenesis, osseointegration and bone ingrowth

Three-dimensional porous titanium alloys printed via electron beam melting have low stiffness similar to that of cortical bone and are promising scaffolds for orthopedic applications. However, the bio-inert nature of titanium alloy is poorly compatible with bone ingrowth. We previously observed that simvastatin/poloxamer 407 thermosensitive hydrogel induces endogenous angiogenic/osteogenic growth factors and promotes angiogenesis and osteogenesis, but the mechanical properties of this hydrogel are poor. The purpose of this study was to construct 3D-printed porous titanium scaffolds (pTi scaffolds) filled with simvastatin/hydrogel and evaluate the effects of this composite on osseointegration, bone ingrowth and neovascularization using a tibial defect rabbit model. Four and eight weeks after implantation, the bone volume, bone mineral density, mineral apposition rate, and push-in maximum force of the pTi scaffolds filled with simvastatin/hydrogel were significantly higher than those without simvastatin (p < 0.05). Moreover, filling with simvastatin/hydrogel significantly enhanced vascularization in and around the pTi scaffolds, and a significant correlation was observed between the volume of new bone and neovascularization (p < 0.01). In conclusion, incorporating simvastatin/poloxamer 407 hydrogel into pTi scaffolds significantly improves neovascularization, osseointegration and bone ingrowth.

A Novel Hybrid Compound LLP2A-Ale Both Prevented and Rescued the Osteoporotic Phenotype in a Mouse Model of Glucocorticoid-Induced Osteoporosis.

Prolonged glucocorticoid (GC) administration causes secondary osteoporosis (GIOP) and non-traumatic osteonecrosis. LLP2A-Ale is a novel bone-seeking compound that recruits mesenchymal stem cells to the bone surface, stimulates bone formation, and increases bone mass. The purpose of this study was to determine if treatment with LLP2A-Ale alone or in combination with parathyroid hormone (PTH) could prevent or treat GIOP in a mouse model. Four-month-old male Swiss-Webster mice were randomized to a prevention study with placebo, GC (day 1-28), and GC+LLP2A-Ale (IV, day 1) or a treatment study with placebo, GC (days 1-56), GC+LLP2A-Ale (IV, day 28), GC+PTH, and GC+LLP2A-Ale+PTH (days 28-56). Mice were killed on day 28 (prevention study) or on day 56 (treatment study). The study endpoints included bone mass, bone strength, serum markers of bone turnover (P1NP and CTX-I) and angiogenesis (VEGF-A), surface-based bone turnover, and blood vessel density. LLP2A-Ale prevented GC-induced bone loss and increased mechanical strength in the vertebral body (days 28 and 56) and femur (day 56). LLP2A-Ale, PTH, and LLP2A-Ale+PTH treatment significantly increased the mineralizing surface, bone formation rate, mineral apposition rate, double-labeled surface, and serum P1NP level on day 56. LLP2A-Ale and PTH treatment increased femoral blood vessel density and LLP2A-Ale increased serum VEGF-A on day 28. Therefore, LLP2A-Ale monotherapy could be a potential option to both prevent and treat GC-induced osteoporosis and bone fragility.

Prolyl hydroxylase domain proteins regulate bone mass through their expression in osteoblasts

The roles of prolyl hydroxylase domain proteins (PHDs) in bone are incompletely understood. Here we deleted the expression of genes encoding PHD1, PHD2, and PHD3 in osteoblasts in mice by breeding the floxed Phd1–3 mice with Col1a1-Cre transgenic mice. Results showed that mice lacking PHD1-3 in osteoblasts (Phd1–3ob−/−) had increased bone mass. Bone parameters such as bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were increased, while trabecular spacing (Tb.Sp) was decreased in Phd1–3ob−/− relative to wild-type (WT) femurs. In contrast, loss of PHD1–3 in osteoblasts did not alter cortical thickness (Cort.Th). The mineralization apposition rate (MAR) was increased in Phd1–3ob−/− bone compared to that of wild-type (WT) bone, demonstrating an enhancement of osteoblast function. Loss of PHD1-3 increased the number of osteoblast progenitors (CFU-OBs) in bone marrow cultures. Interestingly, deleting Phd1–3 genes in osteoblasts increased osteoclast formation in vitro and in bone.

Treatment with hydrogen sulfide attenuates sublesional skeletal deterioration following motor complete spinal cord injury in rats.

Spinal cord injury (SCI)-induced sublesional bone loss represents the most severe osteoporosis and is resistant to available treatments to data. The present study was undertaken to explore the therapeutic potential of hydrogen sulfide (H2S) against osteoporosis in a rodent model of motor complete SCI. SCI was generated by surgical transaction of the cord at the T3-T4 levels in rats. Treatment with NaHS was initiated through intraperitoneal injection of 0.1ml/kg/day of 0.28mol/l NaHS from 12h following the surgery and over 14 subsequent days. H2S levels in plasma of SCI rats were lower, which was restored by treatment with exogenous H2S. Treatment of SCI rats with exogenous H2S had no significant effect on body mass but increased bone mineral density in femurs and tibiae, increased BV/TV, Tb.Th, and Tb.N and reduced Tb.Sp in proximal tibiae, and increased mineral apposition rate (MAR), bone formation rate (BFR), and osteoblast surface and reduced eroded surface and osteoclast surface in proximal tibiae. More importantly, H2S treatment led to a significant enhancement in ultimate load, stiffness, and energy to max force of femoral diaphysis. Treatment of SCI rats with exogenous H2S reduced malondialdehyde (MDA) levels in serum and femurs, decreased hydroxyproline levels, suppressed activities of matrix metallopeptidase 9 (MMP9), and upregulated Wnt3a, Wnt6, Wnt10, and ctnnb1 expression in femurs. Treatment with H2S mitigates SCI-induced sublesional bone loss, possibly through abating oxidative stress, suppressing MMP activity, and activating Wnt/β-catenin signaling.

Osseointegration is improved by coating titanium implants with a nanostructured thin film with titanium carbide and titanium oxides clustered around graphitic carbon

Titanium implants coated with a 500nm nanostructured layer, deposited by the Ion Plating Plasma Assisted (IPPA) technology, composed of 60% graphitic carbon, 25% titanium oxides and 15% titanium carbide were implanted into rabbit femurs whilst into the controlateral femurs uncoated titanium implants were inserted as control. At four time points the animals were injected with calcein green, xylenol orange, oxytetracycline and alizarin. After 2, 4 and 8weeks femurs were removed and processed for histology and static and dynamic histomorphometry for undecalcified bone processing into methylmethacrylate, sectioned, thinned, polished and stained with Toluidine blue and Fast green. The overall bone-implant contacts rate (percentage of bone-implant contacts/weeks) of the TiC coated implant was 1.6 fold than that of the uncoated titanium implant. The histomorphometric analyses confirmed the histological evaluations. More precisely, higher Mineral Apposition Rate (MAR, μm/day) (p<0.005) and Bone Formation Rate (BFR, μm2/μm/day) (p<0.0005) as well as Bone Implant Contact (Bic) and Bone Ingrowth values (p<0.0005) were observed for the TiC coated implants compared to uncoated implants.In conclusion the hard nanostructured TiC layer protects the bulk titanium implant against the harsh conditions of biological tissues and in the same time, stimulating adhesion, proliferation and activity of osteoblasts, induces a better bone-implant contacts of the implant compared to the uncoated titanium implant.

Hyperlipidemia induced by high-fat diet enhances dentin formation and delays dentin mineralization in mouse incisor.

Dyslipidemia has become a serious health problem in children and adolescents worldwide for its high prevalence. Since hard tissues of permanent teeth form mainly during this period and lipids are actively involved in tooth development, the effects of hyperlipidemia on dental tissue formation and mineralization need to be illustrated. In this study, hyperlipidemia model was established in mice fed with high-fat diet (HFD). Micro-CT and histomorphological analyses were performed on the mandibular bones to assess the morphological changes of the mandibular incisor and first molar. After 4weeks of HFD feeding, mice had significantly elevated serum lipid levels compared with mice fed with control diet. After 8weeks, the mandibular incisor presented significantly increased dentin thickness and decreased diameter of pulp cavity in HFD-fed mice compared with control diet-fed mice, while its gross morphology and enamel thickness were not altered. In the mandibular first molar, dentin thickness of root did not show difference between the two groups. Histological section showed that mandibular incisor of HFD-fed mice manifested a wider predentin region and a lower mineral apposition rate compared with that of the control mice. In conclusion, hyperlipidemia induced by HFD feeding enhances dentin formation and delays dentin mineralization in the developing mouse incisor.

Osseointegration behavior of novel Ti-Nb-Zr-Ta-Si alloy for dental implants: an in vivo study.

This study aimed to evaluate the effects of Ti-Nb-Zr-Ta-Si alloy implants on mineral apposition rate and new BIC contact in rabbits. Twelve Ti-Nb-Zr-Ta-Si alloy implants were fabricated and placed into the right femur sites in six rabbits, and commercially pure titanium implants were used as controls in the left femur. Tetracycline and alizarin red were administered 3 weeks and 1 week before euthanization, respectively. At 4 weeks and 8 weeks after implantation, animals were euthanized, respectively. Surface characterization and implant-bone contact surface analysis were performed by using a scanning electron microscope and an energy dispersive X-ray detector. Mineral apposition rate was evaluated using a confocal laser scanning microscope. Toluidine blue staining was performed on undecalcified sections for histology and histomorphology evaluation. Scanning electron microscope and histomorphology observation revealed a direct contact between implants and bone of all groups. After a healing period of 4 weeks, Ti-Nb-Zr-Ta-Si alloy implants showed significantly higher mineral apposition rate compared to commercially pure titanium implants (P < 0.05), whereas there was no significant difference between Ti-Nb-Zr-Ta-Si alloy implants and commercially pure titanium implants (P > 0.05) at 8 weeks. No significant difference of bone-to-implant contact was observed between Ti-Nb-Zr-Ta-Si alloy implants and commercially pure titanium implants implants after a healing period of 4 weeks and 8 weeks. This study showed that Ti-Nb-Zr-Ta-Si alloy implants could establish a close direct contact comparedto commercially pure titanium implants implants, improved mineral matrix apposition rate, and may someday be an alternative as a material for dental implants.

Only minor differences in renal osteodystrophy features between wild-type and sclerostin knockout mice with chronic kidney disease

Renal osteodystrophy affects the majority of patients with advanced chronic kidney disease (CKD) and is characterized by progressive bone loss. This study evaluated the effects of sclerostin knockout on bone in a murine model of severe, surgically induced CKD in both sclerostin knockout and wild-type mice. Mice of both genotypes with normal kidney function served as controls. Tibiae were analyzed using micro-computed tomography, and lumbar vertebrae were analyzed by histomorphometry. Results were tested for statistical significance by 2-way ANOVA to investigate whether bone of the knockout mice reacted differently to CKD compared with bone of wild-type mice. In the tibiae, there was no difference after creation of CKD between wild-type and knockout animals for cortical thickness or cross-sectional moment of inertia. Increases in cortical porosity induced by CKD differed significantly between genotypes in the tibial metaphysis but not in the diaphysis. In the trabecular compartment, no difference in reaction to CKD between genotypes was found for bone volume, trabecular number, trabecular thickness, and trabecular separation. In the lumbar vertebrae, significant differences in response to CKD between wild-type and knockout mice were seen for both bone volume and trabecular thickness. Osteoblast parameters did not differ significantly, whereas osteoclast numbers significantly increased in the wild-type but significantly decreased in knockout mice with CKD. No differences in response to CKD between genotypes were found for bone formation rate or mineral apposition rate. Thus, complete absence of sclerostin has only minor effects on CKD-induced bone loss in mice.

FGF21 Is Not a Major Mediator for Bone Homeostasis or Metabolic Actions of PPARα and PPARγ Agonists.

Results of prior studies suggest that fibroblast growth factor 21 (FGF21) may be involved in bone turnover and in the actions of peroxisome proliferator-activated receptor (PPAR) α and γ in mice. We have conducted independent studies to examine the effects of FGF21 on bone homeostasis and the role of FGF21 in PPARα and γ actions. High-fat-diet-induced obesity (DIO) mice were administered vehicle or recombinant human FGF21 (rhFGF21) intraperitoneally at 0 (vehicle), 0.1, 1, and 3 mg/kg daily for 2 weeks. Additional groups of DIO mice received water or 10 mg/kg rosiglitazone daily. Mice treated with rhFGF21 or rosiglitazone showed expected metabolic improvements in glucose, insulin, and lipid levels. However, bone loss was not detected in rhFGF21-treated mice by dual-energy X-ray absorptiometry (DXA), micro-CT, and histomorphometric analyses. Mineral apposition rate, a key bone formation parameter, was unchanged by rhFGF21, while significantly decreased by rosiglitazone in DIO mice. Bone resorption markers, OPG/RANKL mRNA expression, and histological bone resorption indices were unchanged by rhFGF21 or rosiglitazone. Bone marrow fat was unchanged by rhFGF21, while increased by rosiglitazone. Furthermore, FGF21 knockout mice did not show high bone mass phenotype. Treatment with PPARα or PPARγ agonists caused similar metabolic effects in FGF21 knockout and wild-type mice. These results contrast with previous findings and suggest that FGF21 is not critical for bone homeostasis or actions of PPARα and PPARγ. © 2016 American Society for Bone and Mineral Research.

Repair of segmental radial defects in dogs using tailor-made titanium mesh cages with plates combined with calcium phosphate granules and basic fibroblast growth factor-binding ion complex gel.

Repair of large segmental defects of long bones are a tremendous challenge that calls for a novel approach to supporting immediate weight bearing and bone regeneration. This study investigated the functional and biological characteristics of a combination of a tailor-made titanium mesh cage with a plate (tTMCP) with tetrapod-shaped alpha tricalcium phosphate granules (TB) and basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) to repair 20-mm segmental radial defects in dogs. The defects were created surgically in 18 adult beagle dogs and treated by implantation of tTMCPs with TB with (TB-gel group) or without (TB group) f-IC gel. Each tTMCP fitted the defect well, and all dogs could bear weight on the affected limb immediately after surgery. Dogs were euthanized 4, 8 and 24weeks after implantation. Histomorphometry showed greater infiltration of new vessels and higher bone union rate in the TB-gel group than in the TB group. The lamellar bone volume and mineral apposition rate did not differ significantly between the groups, indicating that neovascularization may be the primary effect of f-IC gel on bone regeneration. This combination method which is tTMCP combined with TB and f-IC gel, would be useful for the treatment of segmental long bone defects.

Miniscrew-assisted slow expansion of mature rabbit sutures.

In this study, we experimentally evaluated whether complex, mature sutures can be separated using skeletal anchorage and light, continuous forces. Twelve adult, 8- to 9-month-old female New Zealand white rabbits were randomly assigned to 1 control group and 2 experimental groups. Open-coil nickel-titanium springs delivered constant forces of 100g across the sagittal suture to miniscrew implants placed bilaterally in the frontal bone. Sutural separation was measured biweekly. Separation was also measured with microcomputed tomography. Bone formation (mineral apposition) was measured with fluorescent labels. Qualitative histologic analyses of the suture tissues were performed using hematoxylin and eosin staining; osteoclasts were evaluated with tartrate resistant acid phosphatase staining. All 24 miniscrew implants remained stable throughout the experiment. There was no statistically significant sutural separation in the control group. In the experimental groups, sutural separation was significant (P<0.05) at all time points after the initial records were taken. The rate of separation was linear during the first 42days. There were moderate correlations (R=0.59-0.89; P<0.05) between miniscrew implant separation and bone marker separation. Mineral apposition rate, which was not measureable in the control group, was significant in the experimental group. The mineral apposition rate was greater between 14 and 28days than between 28 and 38days, and it was greater on the ectocranial than on the endocranial surface. Based on the microcomputed tomography analysis, 3-dimensional sutural volume of the experimental group increased significantly (P=0.02), but surface area did not (P=0.26). It is possible to separate the sagittal suture of mature rabbits. Sutural separation is limited, indicating involvement of other articulations.

Differential Responses to Vitamin D2 and Vitamin D3 Are Associated With Variations in Free 25-Hydroxyvitamin D

25-Hydroxyvitamin D (25D) circulates bound primarily to serum vitamin D binding protein (DBP), with DBP showing higher binding affinity for 25D3 than 25D2. We therefore hypothesized that vitamin D2 (D2) promotes higher serum levels of unbound 25D (free 25D), with different functional responses, relative to vitamin D3 (D3). Week 3 C56BL/6 mice were placed on diets containing either D2 or D3 alone (both 1000 IU/kg). At week 8 and week 16, D2 mice had only 25D2 in circulation (26.6 ± 1.9 and 33.3 ± 4.4 ng/mL), and D3 mice had only 25D3 (28.3 ± 2.0 and 31.7 ± 2.1 ng/mL). At week 8 (44.5 ± 6.4 vs 62.4 ± 11.6 pg/mL, P < .05) and week 16 (78.4 ± 12.6 vs 95.5 ± 11.6), D2 mice had lower serum 1,25-dihydroxyvitamin D relative to D3 mice. By contrast, measured free 25D was significantly higher in D2 mice at week 8 (16.8 ± 0.65 vs 8.4 ± 0.63 pg/mL, P < .001) and week 16 (17.4 ± 0.43 vs 8.4 ± 0.44, P < .001). A two-way ANOVA of bone histomorphometry showed that week 8 D2 mice had significantly higher osteoclast surface/bone surface, eroded surface/bone surface, and mineral apposition rate compared with D3 mice. Osteoblast surface/bone surface was higher in week 8 D2 females but not week 8 D2 males. At week 16, D2 mice had significantly higher bone volume/total volume and trabecular number compared with D3 mice. Differences in bone phenotype were observed despite D2 mice reaching similar serum 25D levels and lower 1,25D levels compared with D3 mice. These data indicate that 25D2 binds less well to DBP than 25D3, with resulting higher levels of free 25D promoting differential effects on bone in mice exposed to D2 alone.

P-glycoprotein overexpression in bone marrow-derived multipotent stromal cells decreases the risk of steroid-induced osteonecrosis in the femoral head.

P‐glycoprotein (P‐gp) plays a role in steroid‐induced osteonecrosis of the femoral head (ONFH), but the underlying mechanism remains unknown. We hypothesized that P‐gp overexpression can prevent ONFH by regulating bone marrow–derived multipotent stromal cell (BMSC) adipogenesis and osteogenesis. BMSCs from Sprague–Dawley rats were transfected with green fluorescent protein (GFP) or the multidrug resistance gene 1 (MDR1) encoding GFP and P‐gp. Dexamethasone was used to induce BMSC differentiation. Adipogenesis was determined by measuring peroxisome proliferator‐activated receptor (PPAR‐γ) expression and the triglyceride level. Osteogenesis was determined by measuring runt‐related transcription factor 2 (Runx2) expression and alkaline phosphatase activity. For in vivo experiments, rats were injected with saline, BMSCs expressing GFP (GFP‐BMSCs) or BMSCs expressing GFP‐P‐gp (MDR1‐GFP‐BMSCs). After dexamethasone induction, adipogenesis was determined by measuring PPAR‐γ expression and fatty marrow, whereas osteogenesis was detected by measuring Runx2 expression, trabecular parameters and the mineral apposition rate, followed by evaluation of the incidence of ONFH. Overexpression of P‐gp in BMSCs resulted in markedly decreased expression of adipogenic markers and increased expression of osteogenic markers. Compared with rats injected with saline, rats injected with GFP‐BMSCs showed reduced ONFH, and the injected GFP‐positive BMSCs attached to trabecular surfaces and exhibited an osteoblast‐like morphology. Compared with the rats injected with BMSCs expressing GFP alone, rats injected with BMSCs overexpressing GFP and P‐gp showed lower adipocytic variables, higher osteogenic variables and lower incidence of ONFH. Overexpression of P‐gp inhibited BMSC adipogenesis and promoted osteogenesis, which reduced the incidence of steroid‐induced ONFH.

A neoflavonoid dalsissooal isolated from heartwood of Dalbergia sissoo Roxb. has bone forming effects in mice model for osteoporosis

Dalbergia sissoo Roxb. is a well known medicinal plant of India, enriched with various flavonoids used for treating multiple diseases. Earlier, we have shown that extract of Dalbergia sissoo Roxb. leaves mitigate ovariectomy induced bone loss and pure compounds (neoflavonoids) isolated from it, promote osteoblastogenesis in primary calvarial osteoblasts cells in vitro. Here, we hypothesize that dalsissooal (DSL), a novel neoflavonoid isolated from the heartwood of Dalbergia sissoo Roxb. is an important constituent of the extract that imparts bone forming effects. Treatment with DSL enhanced trabecular bone micro-architecture parameters, biomechanical strength, increased bone formation rate and mineral apposition rate in OVx mice comparable to 17β-estradiol. It increased bone formation by enhancing osteoblast gene expression and reduced bone turnover by decreasing osteoclastic gene expressions. Interestingly, we observed that DSL has no uterine estrogenic effects. At cellular levels, DSL promoted differentiation of bone marrow cells as well as calvaria osteoblast cells towards osteoblast lineage by enhancing differentiation and mineralizing ability to form mineralizing nodules via stimulating BMP-2 and RunX-2 expressions. Overall, our data suggest that oral supplementation of a novel neoflavonoid dalsissooal isolated from heartwood of Dalbergia sissoo Roxb. exhibited bone anabolic action by improving structural property of bone, promoting new bone formation and reducing bone turnover rate in post-menopausal model for osteoporosis with no uterine hyperplasia.

(18)F-fluoride-PET for dynamic in vivo monitoring of bone formation in multiple myeloma.

BackgroundBone disease in multiple myeloma is characterized by reduced bone formation. The gold standard of bone formation is the mineral apposition rate (MAR), an invasive technique reflecting bone formation at a single site. We compared 18F-fluoride-PET with the MAR in myeloma patients.MethodsBone formation was measured before and after bortezomib treatment by determination of the MAR in iliac bone marrow biopsies and the measurement of 18F-uptake.ResultsThe inter- and intra-individual variations in 18F-uptake (SUVA50%) were pronounced as 33.50 (range 4.42 to 37.92) and 27.18 (range 4.00 to 31.18), respectively. A significant correlation between the MAR and 18F-uptake was found (r = 0.80, p = 0.017). There was a heterogeneous response after treatment varying from −2.20 to 4.53.ConclusionsIliac 18F-uptake was associated with the local MAR in myeloma patients. Furthermore, 18F-fluoride-PET demonstrated the heterogeneity of in vivo bone formation, enabling monitoring during treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0197-4) contains supplementary material, which is available to authorized users.

Bone Formation is Affected by Matrix Advanced Glycation End Products (AGEs) In Vivo.

Advanced glycation end products (AGEs) accumulate in bone extracellular matrix as people age. Although previous evidence shows that the accumulation of AGEs in bone matrix may impose significant effects on bone cells, the effect of matrix AGEs on bone formation in vivo is still poorly understood. To address this issue, this study used a unique rat model with autograft implant to investigate the in vivo response of bone formation to matrix AGEs. Fluorochrome biomarkers were sequentially injected into rats to label the dynamic bone formation in the presence of elevated levels of matrix AGEs. After sacrificing animals, dynamic histomorphometry was performed to determine mineral apposition rate (MAR), mineralized surface per bone surface (MS/BS), and bone formation rate (BFR). Finally, nanoindentation tests were performed to assess mechanical properties of newly formed bone tissues. The results showed that MAR, MS/BS, and BFR were significantly reduced in the vicinity of implant cores with high concentration of matrix AGEs, suggesting that bone formation activities by osteoblasts were suppressed in the presence of elevated matrix AGEs. In addition, MAR and BFR were found to be dependent on the surrounding environment of implant cores (i.e., cortical or trabecular tissues). Moreover, MS/BS and BFR were also dependent on how far the implant cores were away from the growth plate. These observations suggest that the effect of matrix AGEs on bone formation is dependent on the biological milieu around the implants. Finally, nanoindentation test results indicated that the indentation modulus and hardness of newly formed bone tissues were not affected by the presence of elevated matrix AGEs. In summary, high concentration of matrix AGEs may slow down the bone formation process in vivo, while imposing little effects on bone mineralization.

TGF-β Signaling Regulates Cementum Formation through Osterix Expression

TGF-β/BMPs have widely recognized roles in mammalian development, including in bone and tooth formation. To define the functional relevance of the autonomous requirement for TGF-β signaling in mouse tooth development, we analyzed osteocalcin-Cre mediated Tgfbr2 (OCCreTgfbr2fl/fl) conditional knockout mice, which lacks functional TGF-β receptor II (TβRII) in differentiating cementoblasts and cementocytes. Strikingly, OCCreTgfbr2fl/fl mutant mice exhibited a sharp reduction in cellular cementum mass with reduced matrix secretion and mineral apposition rates. To explore the molecular mechanisms underlying the roles of TGF-β signaling through TβRII in cementogenesis, we established a mouse cementoblast model with decreased TβRII expression using OCCM-30 cells. Interestingly, the expression of osterix (Osx), one of the major regulators of cellular cementum formation, was largely decreased in OCCM-30 cells lacking TβRII. Consequently, in those cells, functional ALP activity and the expression of genes associated with cementogenesis were reduced and the cells were partially rescued by Osx transduction. We also found that TGF-β signaling directly regulates Osx expression through a Smad-dependent pathway. These findings strongly suggest that TGF-β signaling plays a major role as one of the upstream regulators of Osx in cementoblast differentiation and cementum formation.